1. Field of the Invention
This invention is directed to cells and methods for screening and propagating Ehrlichi chaffeensis and Ehrlichia canis.
2. Background Art
Human ehrlichiosis and Rocky Mountain spotted fever are both tick-borne diseases that cause significant morbidity and mortality in the United States (12, 14, 17, 19). Approximately 260 cases of human ehrlichiosis, including 5 fatalities, have been serologically confirmed in the United States since 1986 (3). In addition, one case each has been reported in Portugal (16) and Mali, Africa (18). In contrast, state health departments reported 649 cases of Rocky Mountain spotted fever in 1990 (4). Rocky Mountain spotted fever is a reportable disease, human ehrlichiosis is not. Therefore, reliable estimates of the number of human ehrlichiosis cases are not available.
Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis, was first isolated in 1990 (2, 8). Prior to the isolation of E. chaffeensis, human ehrlichiosis was diagnosed based upon a fourfold change in antibody titer to E. canis (6, 7). E. canis, the etiologic agent of canine ehrlichiosis, is closely related to E. chaffeensis and was originally suspected of being the etiologic agent of human ehrlichiosis (15).
Human ehrlichiosis is generally characterized by fever, malaise, headache, myalgia, rigor, nausea/vomiting, arthralgia, rash, and cough (10, 11). Many of the same clinical signs and symptoms characterize an infection with Rickettsia rickettsii, etiologic agent of Rocky Mountain spotted fever (19). Although the characteristic rash of Rocky Mountain spotted fever, on the palms of the hands and the soles of the feet, may help to distinguish this disease from human ehrlichiosis, the rash is often not observed during the first few days after onset of illness (19). If a febrile illness after tick exposure is reported, neither disease can be immediately ruled out.
R. rickettsii are short rods 0.3-0.5 .mu.m in diameter and 0.8-2.0 .mu.m in length. Growth occurs in the cytoplasm, sometimes in the nucleus, of certain vertebrate and arthropod cells. Ultrathin sections viewed by electron microscopy reveal typical envelopes consisting of cell wall and cytoplasmic membranes and internal structures analogous to the ribosomes and DNA strands identified in other microorganisms.
E. chaffeensis are often pleomorphic, coccoid to ellipsoidal. Individual organisms are approximately 0.5 .mu.m in diameter, and morulae range in size up to 4.0 .mu.m in diameter. These organisms occur in membrane-bound vacuoles in the cytoplasm of leukocytes, forming inclusions that contain variable numbers of organisms. Members of the tribe Ehrlichieae have distinct ribosomes and DNA strands. Clumps of ribosomes are homogenously distributed in the cytoplasm rather than marginated beneath the cytoplasmic membrane. Compared with those of the rickettsiae or ordinary bacteria, the DNA and ribosomes in members of the tribe Ehrlichieae are more loosely packed in the cytoplasm.
R. rickettsii organisms enter by tick bite, spread via the lymphatics and blood stream to all parts of the body including skin, brain, lungs, kidneys, heart, liver, spleen, pancreas, and gastrointestinal tract (19). In each tissue, the rickettsiae proliferate within endothelial cells and vascular smooth muscle cells (19). R. rickettsii can be cultivated in a variety of cell lines including Vero cells, L cells, primary chicken embryo fibroblasts, WI-38, and HeLa. However, R. rickettsii must be maintained for several weeks, because the incubation period of rickettsiae can be long and growth with new isolates may be sparse for a week or longer (19).
Little is known about the pathogenesis of E. chaffeensis. However, in one fatal case, Ehrlichia sp. organisms were demonstrated by immunohistology in the splenic cords and sinuses, splenic periarteriolar lymphoid sheaths, hepatic sinusoids, lymph nodes, lung microvasculature, bone marrow, kidney, and epicardium (9). Ehrlichia sp. morulae were most frequently detected in large mononuclear cells and infrequently in small lymphocyte-like cells (9).
Since the quantity of an acute blood specimen received is often limited and successful isolation is directly related to the volume of blood inoculated, there is a need for one cell line that would support the growth of both E. chaffeensis and R. rickettsii. This invention satisfies this need.